Relating to hepatitis B vaccine

ABSTRACT

Immunogenically active fragments of hepatitis B surface antigen, which are a glycoprotein of molecular weight about 28,000 (gp 28) and a protein of molecular weight about 23,000 (p 23), obtained by known methods by treatment of surface antigen with detergent, are prepared in micelle form substantially free from detergent. The micelles are at least as immunogenic as the unfragmented antigen from which they are derived and are useful in the preparation of hepatitis B vaccine. The detergent is removed by layering the detergent containing fragmentation product on an aqueous buffer containing a sucrose gradient and centrifuging the layered buffer when micelles containing both gp 28 and p 23 form and can be recovered in substantially detergent free form from the centrifuged layered buffer.

This is a continuation of application Ser. No. 614,714 filed May 29,1984, now U.S. Pat. No. 4,554,157 (which is a continuation of Ser. No.341,142, filed Jan. 20, 1982 [abandoned]).

DESCRIPTION

This invention relates to hepatitis B vaccine and is particularlyconcerned with a process for the production of hepatitis B vaccine fromfragments of hepatitis B surface antigen.

Virus hepatitis is a major public health problem in all parts of theworld. The infection is caused by at least four different viruses ofwhich hepatitis A and hepatitis B have been identified andcharacterised. A third form of hepatitis has recently been recognisedand is believed to be caused by two viruses different from one anotherand from hepatitis A and B. Hepatitis B affects every field of medicalpractice throughout the world and it is the long-term persistence ofthis virus which causes particular problems, for example in the bloodtransfusion service. Hepatitis B virus can be transmitted directlythrough the skin so that the possible modes of entry are numerous.

The need for a vaccine to combat hepatitis B infection has beenrecognized for many years but traditional methods of vaccine production,by attenuation of the pathogenic virus by repeated passage throughtissue culture have not proved possible because of the difficulty ofgrowing this particular virus under artificial conditions. Onealternative approach that has led to limited success in vaccineproduction has been the isolation and use of hepatitis B surface antigenalone. This surface antigen is essentially the protein coat of the viruswhich are usually spheres of protein 22 nm across. Experimental vaccineshave been prepared from the plasma of healthy carriers of hepatitis Bvirus and it has been found that such a vaccine is capable of producingprotective surface antibody and has led to protection in chimpanzeetests and in limited clinical trials.

One of the practical problems associated with the use of viral vaccinesprepared from plasma is that, in addition to containing the desiredantigenic material which provokes the production of the desiredprotective antibodies, the plasma derived vaccine contains numerousother antigenic materials which are all capable of provoking theproduction of other antibodies and the presence of such contaminatingantigenic materials can lead to undesirable side-effects.

In an attempt to avoid the problems associated with these contaminatingantigenic materials, attempts have been made to modify hepatitis Bsurface antigen (HBsAg) by breaking down the surface antigen intopolypeptide fragements. Essentially, the fragementation of HBsAg wasachieved by treating the HBsAg with the non-ionic detergent Triton X-100by overnight incubation at 37° C. in the presence of a pH 7.3 buffer.The fragmented HBsAg was then passed through a Sepharose column so thatthe various fragments could be separated from one another. The molecularweight of the resulting fragments were determined by sedimentationtechniques and the immunological properties of the resulting fragmentswere also examined. These tests showed that two of the resulting peptidefragments had significant hepatitis B surface antigen activity. Thesewere a glyco polypeptide fragment having a molecular weight of about28,000 and a polypeptide fragment having a molecular weight of about23,000. This fragmentation of HBsAg has been described in more detail bySkelly, Howard and Zuckerman in J.Gen. (1979) 44 679-689.

While numerous tests were carried out on the glyco polypeptide fragmentof molecular weight 28,000 and the polypeptide fragment of molecularweight 23,000 obtained as described above, to compare the immunogenicityof these fragments with the HBsAg from which they were derived, nosatisfactory method was then available for the removal of all traces ofthe detergent which was used in the disruption of HBsAg andconsequently, the fragments obtained by the procedure described in theSkelly et al paper mentioned above were unsuitable for the production ofa clinically acceptable vaccine.

We have now found a method by which it is possible to removesubstantially all traces of detergent from the immunogenically activefragments obtained by detergent disruption of HBsAg so that a clinicallyuseful vaccine can be prepared.

Accordingly, the present invention provides a process for the productionof a detergent free protein fraction, suitable for use in theformulation of a vaccine against hepatitis B virus infection, whichcomprises treating hepatitis B surface antigen with a non-ionicdetergent to form a polypeptide mixture including an immunogenic glycopolypeptide fragment of molecular weight about 28,000 and an immunogenicpolypeptide fragment of molecular weight about 23,000, introducing thepolypeptide mixture to form a layer on top of an aqueous solutionbuffered to a pH which avoids denaturation of the glyco polypeptide ofmolecular weight about 28,000 and the polypeptide of molecular weightabout 23,000, said aqueous buffer containing sucrose in a concentrationgradient of at least 20% to not more than 65%, preferably not more than50%, weight/volume, centrifuging the layered buffer and recovering fromthe upper part of the resulting buffer an aqueous fraction substantiallyfree from detergent and containing micelles of the glyco polypeptide ofmolecular weight about 28,000 and the polypeptide of molecular weightabout 23,000.

The process of the present invention may be used in relation tohepatitis B surface antigen of any origin e.g. of human or of chimpanzeeorigin.

The detergent used to treat the surface antigen will usually be analkylaryl polyether alcohol such as one of the Tritons.

The process of the present invention preferably involves the use of a20-50% or 20-60% w/v linear sucrose gradient in the aqueous buffer sinceit has been found that the use of such an aqueous buffer in which thesucrose concentration is stepped up in a linear manner across the range20% w/v-50% or 60% w/v permits a substantially complete separation ofthe detergent from the immunogenically active glyco polypeptide ofmolecular weight about 28,000 hereinafter designated gp 28 and thepolypeptide of molecular weight about 23,000 hereinafter designated p23. The sucrose gradient technique also permits the generation ofmicelles from gp 28 and p 23. Such substantially detergent-free aqueouspreparations avoid the problems of the known detergent containingmaterials from the clinical point of view and, furthermore, avoid theside-effect problems associated with the use of plasma-derived materialscontaining whole hepatitis B surface antigen.

The process of the present invention utilises an aqueous buffer whichwill maintain the pH of the aqueous medium at a value which avoidsdenaturation of gp 28 and p 23. As with most immunogenically activematerials, the working pH range is about 4 to 8 and satisfactory resultshave been obtained with a pH of about 7.0 to 7.5. So-called TNE buffer,based on sodium phosphate, sodium chloride and ethylene diaminetetraacetic acid and having a pH of 7.4 has been found to be quitesuitable for use in the present invention.

The layered sucrose gradient is centrifuged in accordance with thepresent invention to separate the detergent from the desired gp 28 and p23 fragments. The exact speed of centrifugation is not critical but, inorder to bring about separation in a reasonable period of time, we findit convenient to use speeds corresponding to a force of about 40 g to250,000 g and usually, speeds corresponding to a force of at least200,000 g will be used.

The time for which the centrifugation is carried out is again notcritical but will depend upon the speed of rotation in the centrifuge,an adequate degree of separation of the desired immunogenic material anddetergent normally being achieved within about six hours. To ensure thatthe detergent removal is as complete as possible, centrifugation willnormally be carried out for longer than six hours and, as a practicalmatter, the centrifugation will normally be carried out for up to 24 orperhaps 48 hours.

In order to ensure uniformity of results, it is desirable to control thetemperature during the centrifugation step although the particulartemperature selected is not critical to the separation. There isnormally no advantage to be gained by increasing the temperature abovethe ambient room temperature although, under some circumstances, it maybe desirable to operate at slightly below room temperature. A practicalworking range is 4° to 20° C. although operation above 20° C. or below4° C. is possible in accordance with the invention.

The process of the present invention produces a detergent free aqueousmaterial, suitable as a clinical vaccine, containing micelles of gp 28and p 23. To the best of our knowledge and belief, such detergent freeaqueous preparations obtained by our procedure are new. Detergent freeaqueous solutions containing micelles of gp 28 and p 23 and othermaterial immunogenically similar to hepatitis B surface antigen derivedfrom any other source such as cell lines or by genetic manipulation orsynthesised chemically form a further aspect of the present invention.

The detergent free gp 28 and p 23 fragments may be formulated intoclinical vaccines by methods known per se. For example, the immunogenicmaterial may be formulated in a pyrogen free aqueous carrier containingabout 5 to 50 μg per milliliter of the immunogenic material and suchaqueous vaccines may also incorporate conventional vaccine adjuvantssuch as aluminum hydroxide or one of the organic adjuvants.

Vaccines containing the detergent free gp 28 and p 23 fragments obtainedin accordance with the present invention have been found to be effectivein both in vitro tests and in vivo tests in guinea pigs, mice andchimpanzees. The chimpanzee tests have shown that not only do vaccinesobtained by the present invention generate high antibody titres inchimpanzees but also that they give an acceptable measure of protectionwhen the immunised chimpanzee is challenged by the introduction ofpathogenic virus into the animal.

The following Examples are given to illustrate the invention.

EXAMPLE 1

Hepatitis B surface antigen, obtained from the plasma of an infectedchimpanzee, was purified in the form of 20 to 25 nm particles by theprocedure described by Skelly et al (1978) J. Gen. Virol. 41 447-457.The resulting HBsAg was disrupted by overnight incubation at 37° C. inthe presence of Triton X-100 and NaCl at final concentrations of 2% w/vand 0.5 M respectively as described in Skelly et al J. Gen. Virol.(1979) 44, 679-689. The solubilised material was passed through a columnof concanavalin A Sepharose equilibrated in the complete disruptionbuffer supplemented with 1M CaCl₂ and MnCl .sub.. The fraction bound bythe column was eluted with α-methylmanoside and was found to be theglyco polypeptide of molecular weight about 28,000 (gp 28) and thepolypeptide of molecular weight about 23,000 (p 23).

The gp 28 and p 23 material were then separated from the detergent bylayering on top of a linear sucrose gradient. The aqueous buffer usedwas a PNE buffer (0.05M tris HCl, 0.14M NaCl and 0.001M ethylene diaminetetraacetic acid) having a pH of 7.4. A linear sucrose concentrationgradient was established in this aqueous buffer ranging from 20% w/v to50 w/v. The detergent containing sample of gp 28 and p 23 was thenintroduced on top of the sucrose containing buffer and the liquid wasthen centrifuged in a Beckman SW40 centrifuge at 220,000 g for 24 hoursat 4° C. 0.5 ml fractions were then collected from the top of the tubeand the position of the polypeptide monitored by gamma spectrometry.Tests using iodine 125 labelled polypeptides showed that there was apeak approximately two-thirds of the way down the gradient. However, incontrol experiments in which the buffer contained 2% Triton the wholeway through, the radioactivity remained at the top of the gradientshowing that the use of a detergent free sucrose gradient was aneffective way of separating the gp 28 and p 23 material from thedetergent. The fact that the radioactivity peak was two-thirds of theway down the gradient, when the detergent free sucrose gradient was usedin accordance with the invention shows that the gp 28 and p 23 materialsreassociate to form polypeptide micelles which have an increasedsedimentation coefficient compared to the immunogenic material in thedetergent containing starting material.

Samples of the Triton free gp 28 and p 23 were incorporated into anaqueous buffer of the type described above in which a linear gradient ofcaesium chloride had been established at a concentration of 1.1 to 1.4grams/cm³. This material was centrifuged in the same Beckman centrifugeat 22,000 g for 24 hours at 4° C. and 0.5 ml fractions were thenrecovered from the top of the tube. Buoyant density determinations weremade using an Abbe type refractometer which showed that the buoyantdensity was 1.25 g/ml compared with a density of 1.19 g/ml for intactHBsAg particles. Electron microscopy showed that the micelles of gp 28and p 23 were pleomorphic and fluffy in appearance with a mean diameterof 120 nm. The micelles could be aggregated by rapid anti-HBs.Polyacrylamide gel electrophoresis showed that both gp 28 and p 23 werepresent in the micelles in the same proportion as in the detergentsolution indicating that the micelles were not formed preferentiallyfrom one or other of the polypeptides.

RECOVERY OF SEROLOGICAL ACTIVITY

Specific HBsAg activity was monitored by reverse passivehaemagglutination (RPHA) on the gp 28 and p 23 material and also on theHBsAg material after disruption with Triton and on the eluate from theCon-A-Sepharose. In one experiment in which 10 mg of HBsAg wasdisrupted, 3.4 mg was recovered from the Con-A-Sepharose column and 2.5mg protein was recovered from the sucrose gradient representing 73% ofthe amount of gp 28 and p 23 originally present. The RPHA titre permilligram of protein was found to be 2.2 ×10³ for the protein micellesformed in the sucrose gradient compared to 3.7×10⁵ for the eluate fromthe Sepharose and 9.9×10⁴ for the HBsAg after disruption with Triton.

IMMUNOGENICITY OF THE MICELLES OF GP 28 AND P 23

The immunogenic properties of the micelles obtained by the presentinvention and of the intact 22 nm HBsAg particles from which themicelles were obtained, were compared using a mouse potency test. Thetwo preparations under test were first dialysed against a 0.1M phosphatebuffer (PBS) of pH 6.6 and then made up to a final concentration of 200μg protein per ml. In this phosphate buffer, 1 ml of a 2% suspension ofthe aluminum phosphate was added to 6 ml of each sample and absorptionof the protein was carried out by mixing the suspensions overnight. Thealuminum phosphate was then centrifuged and washed once with PBS.Measurements of protein and serological activity by reverse passivehaemagglutination of the supernatants showed that more than 95% of theantigen had been absorbed on aluminum phosphate. The precipitate wasthen resuspended in PBS to give a final aluminum phosphate concentrationof 0.04%. The suspensions were then diluted approximately with 0.4%aluminum phosphate in PBS in order to make both the volume and thealuminum phosphate content of the inocula the same for all antigendoses. Batches of 6 mice per dose were injected intraperitoneally with20, 10 or 5 μ g of either the intact 22 nm particle or the micelle of gp28 or p 23 material in 100 μl volumes. Two booster doses were given atweekly intervals. The mice were bled 11 days after the last inoculationand the titres of antibody in the sera determined.

FIG. 1 of the accompanying drawings show protein A-radioimmunoassay ofanti-HBs in sera of mice inoculated with three 10 μg doses of either themicelle formulation of the invention (shown as •) or a formulation ofthe intact 22 nm particle HBsAg (shown as o). The sera were initiallydiluted 1:10 in phosphate buffer saline (PBS) containing 0.5% bovineserum albumen (BSA) and serial two-fold dilutions were mixed with ¹²⁵I-HBsAg (the intact 25 nm particles, 10 μl 24,000 cpm) and incubatedovernight at room temperature. Protein A Sepharose (5% in PBS-BSA) wasthen added and the samples shaken vigorously 1 hour at room temperature.The Sepharose was pelleted by low speed centrifugation and the pelletsand supernatant separated prior to counting. The points on FIG. 1 showthe mean counts per minute associated with the precipitated immunecomplexes from six sera and the vertical lines show the standarddeviation.

As shown in FIG. 1 there was consistently higher antibody levels in allthe mice inoculated with the micelle preparation at the test dose level.Similar high antibody level were observed with other dose levels of themicelle preparation.

EXAMPLE 2

Surface antigen was isolated as 22 nm particles from persistentlyinfected chimpanzee serum by the Skelly et al (1978) method mentioned inExample 1 and was disrupted substantially as described in Example 1using slight modifications from the Skelly et al (1979) procedurementioned. The 0.01M-tris-HCl pH 7.3 buffer contained a final TritonX-100 concentration of 2% w/v and the supplements in the buffer used forequilibration of the Concanavalin A Sepharose were 1mM CaCl₂ and1mM-MnCl₂. The suspension was gently mixed on a rotator for 60 minutesand then packed in a 1×10 cm column and washed with buffer to removeunbound material. Bound material was eluted with 0.01 M-tris-HCl, pH 7.3containing 2% TX-100, 0.5M NaCl and 5% α-methyl-D-mannoside (α-mm). Peakfractions were then centrifuged on 20-60% w/v linear sucrose gradients(free from detergent) in TNE buffer (0.5 M tris-HCl, pH 7.4, 0.14 MNaCl, 0.001 M EDTA) at 220,000 g for 24 hours at 15° C. in a BeckmanSW40 rotor. The position of the desired gp 28 and p 23 fractions in thesucrose gradient was identified as explained in Example 1 andTriton-free samples of gp 28 and p 23 in micelle form were recovered.

Buoyant density measurements were carried out as described in Example 1and was found to be 1.22 g ml⁻¹ compared to 1.19 g ml⁻¹ for theunfragmented surface antigen.

Polyacrylamide gel electrophoresis of the micelles shows that theycontain essentially the gp 28 and p 23 polypeptides in the sameproportions as the unfragmented surface antigen. Electron microscopyshows the micelles to be roughly spherical in shape and of diameters140-250 nm. Measurement of over 200 particles indicates an averagediameter of 180 nm.

Specific hepatitis B surface antigen activity was monitored by eitherreverse passive haemagglutination (RPHA) orradioimmunoassay (RIA)throughout the detergent removal steps and no loss of serologicalactivity was detected either after disruption with the Triton orfollowing elution from the Con-A Sepharose. An increase in specificactivity has been noticed, perhaps as a result of the removal of newimmunoreactive constituents present in the original particle, and atleast 70% of the original antigenicity is retained in the micellepreparation. This micelle preparation meets the World HealthOrganisation requirements in the mouse potency assay test and in thechimpanzee safety and protective tests.

EXAMPLE 3

The procedure described in Example 2 was repeated but using 22 nmparticles of surface antigen recovered by the Skelly et al (1978)procedure from the serum of a human carrier. A substantially similarmicelle product was obtained as that described in Example 2 except thatthe buoyant density was found to be 1.24 g ml⁻¹ and the micelles werefound to be slightly larger (mean diameter 200 nm) compared to thechimpanzee originating material.

I claim:
 1. A process for producing a protein fraction suitable for usein the formulation of a vaccine against Hepatitis B virus infectionwhich includes the steps of(1) forming an aqueous preparation comprisingnon-ionic detergent and an immunogenic polypeptide obtained either bychemical synthesis or from a genetically engineered host cell and beingimmunogenically similar to the mixture of polypeptide of molecularweight about 23,000 and glycopolypeptide of molecular weight about28,000 obtained by treating serum-originating particles of diameter 20to 25 nm bearing Hepatitis B surface antigen with a non-ionic detergentfollowed by affinity chromatography, (2) introducing said aqueouspreparation to form a layer on top of an aqueous solution buffered to apH which avoids denaturation of the polypeptide, said aqueous buffercontaining sucrose in a concentration gradient of at least 20% to notmore than 65% weight/volume, (3) centrifuging the layered buffer andrecovering from the buffer an immunogenic aqueous fraction substantiallyfree from detergent and containing micelles of the immunogenicpolypeptide.
 2. A process according to claim 1 wherein the concentrationgradient of the sucrose is at least 20% but not more than 50%weight/volume.
 3. A process according to claim 1 wherein theconcentration gradient of the sucrose is a linear gradient increasingfrom 20% up to 50% or 20% up to 60% weight/volume.
 4. A processaccording to claim 1 wherein the sucrose containing aqueous buffer has apH of 7.0 to 7.5.
 5. A process according to claim 1 wherein the sucrosecontaining aqueous buffer includes sodium phosphate, sodium chloride andethylene diamine tetra-acetic acid.
 6. A process according to claim 1wherein the detergent containing polypeptide preparation is introducedas a layer on top of the sucrose containing aqueous buffer and thelayered buffer centrifuged at a speed corresponding to a force of 40 to250,000 g.
 7. A process according to claim 6 wherein the speedcorresponds to a force of at least 200,000 g.
 8. A process according toclaim 7 wherein the centrifugation is carried out for at least 6 hoursat 4° to 20° C.
 9. A process according to claim 8 wherein thecentrifugation is carried out for about 24 hours.
 10. A processaccording to claim 1 wherein detergent-free micelles of polypeptide arerecovered from the centrifuged layered buffer and formulated in apyrogen-free aqueous medium.
 11. A process according to claim 1 whereinthe detergent is an aralkyl polyether alcohol.